The biogenesis of integral membrane proteins is an essential and complex process. Until recently, the targeted delivery of an important class, tail-anchored (TA) membrane proteins, was poorly understood. TA-proteins are defined topologically as containing a single trans-membrane domain (TM) near the C-terminus. This constraint prevents them from following the canonical signal recognition particle dependent co-translational targeting pathway. TA-proteins are involved in many key cellular processes including protein localization, vesicular trafficking, regulation of apoptosis and viral assembly and have been linked to a number of diseases. They are found in most eukaryotic membranes;however, they are initially delivered to either the endoplasmic reticulum (ER) or the mitochondrial outer membrane. TA-proteins are targeted to the ER by the newly discovered Get pathway (Guided entry of TA-proteins). In yeast, the central component of this pathway is the targeting factor Get3, a universally conserved ATPase that binds specifically to the TM of a TA-protein and uses ATP hydrolysis to deliver the TA-protein to the ER membrane. At the membrane, two proteins, Get1 and Get2, are thought to act as receptors for Get3 to ensure proper targeting and release of the TA-protein from Get3. Upstream of Get3, the proteins Get4, Get5 and Sgt2 are thought to mediate delivery to Get3 and may play a role in discriminating alternate delivery pathways. The identification of proteins in this pathway has provided an early framework for the targeting model. Recently, key structural work by our group, along with efforts by others, has begun to shed light on this process. Our early work has primed us to fully elucidate the roles of each component in the system. In the following proposal we describe how we plan to characterize each step in targeting both structurally and biochemically. The aims are to 1) determine the role of conformational changes and nucleotide hydrolysis in TA-protein targeting by Get3, 2) understand the process of discriminating TA- substrates and their delivery to Get3 and 3) elucidate the route of insertion of a TA-protein into a membrane. PUBLIC HEALTH RELEVANCE: Tail-anchor (TA) proteins, linked to a number of diseases such as cancer, are a diverse class of integral membrane proteins with key roles in many cellular processes. The broad importance of these proteins makes it critical that there is a detailed understanding of their biogenesis. The program proposed here aims to elucidate the mechanistic details of the biological principles that govern the synthesis of TA-proteins potentially leading to novel therapeutic strategies. !